Genetic correction of CF will require the development of vectors capable of achieving direct, in vivo gene transfer to the cells of the airway epithelia. Molecular conjugate vectors offer may potential advantages for this application. The present proposal will evaluate the feasibility of gen delivery to CF respiratory epithelia employing molecular conjugate vectors towards the ultimate goal of achieving gene therapy for CF. One Specific Aim of this proposal is thus to determine if gene transfer can be accomplished to CF airway epithelial cells by the receptor-mediated endocytosis pathway via conjugate vectors utilizing a targetable cell surface receptor. These studies will utilize primary cultures of human airway epithelial cells. To achieve genetic correction by direct in vivo delivery, conjugates must accomplish efficient gene transfer to target cells. Since it is known that gene transfer mediated by molecular conjugates can be dramatically augmented by agents that cause target cell endosomolysis, the second Specific Aim of this proposal is to devise conjugate vectors of higher efficiency by the incorporation of a specific endosomolysis mechanism into the conjugate design. The strategy employed will focus on utilization of adenoviral-mediated endosomolysis by identification of the adenovirus endosomolysis principle residing within the capsid structure. The selective incorporation of this agent would allow employment of viral entry functions without the biosafety limitations inherent in recombinant viral vectors. As it may be a physiologic constraint to selectively express CFTR within a defined airway epithelial cell subset, the third Specific Aim of this proposal is the design of molecular conjugate vectors with the capacity to target these cells. This strategy will capitalize on the cell-specific tropism of influenza virus and Mycoplasma pneumoniae for ciliated respiratory epithelial cells. The cell binding domains of these agents will be prepared by recombinant techniques and incorporated into conjugate design to yield a molecular conjugate vector with a corresponding specificity. In summary, the present proposal seeks to: 1) demonstrate the feasibility of conjugate-mediated gene delivery to airway epithelial cells; 20 confer upon such a conjugate the higher efficiency gene transfer capacity required for in vivo delivery; and 3) target these conjugates to achieve cell-specific delivery to a physiologically relevant airway epithelial cells subset. Achievement of these Aims will potentially yield a vector system with the features required to achieve gene therapy for CF.